Li, Kelly; Li, Jisheng; Ji, Hong; Patel, Sunali; Hayashibara, Kathleen; Fantin, Nicole; Hartshorne, Toinette; Pagani, Ioanna; Zeringer, Emily; Puri, Nitin and Varma, Kamini Clinical R&D, Genetic Science Division, Life Science Group, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080

Performance Verification of the Comprehensive Respiratory Tract Microbiota (RTM) Panel Using QCMD Control Samples And Repository Specimen

For research use only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific. All rights reserved.

INTRODUCTION

As one of the most common infections in humans, a respiratory infection is one high risk factor in pediatric, geriatric and immuno-comprised populations. Identification of the type of infection and pathogen(s) involved could help understanding the pathogenesis, severity and outcome of the infection. Currently, several respiratory panels are available in the market that utilize different technologies. To meet the needs of a wider respiratory pathogen coverage, higher sample throughput and content customization capability and lower cost, we developed a respiratory tract microbiota (RTM) panel that detects a total 42 pathogens, including viral, bacterial and fungal microorganisms on a nanofluidic device that can simultaneously interrogate all the targets on the panel via spatial multiplexing. After analytical performance, we also evaluated the panel with a large numbers of repository samples and Quality Control and Proficiency Test samples. These samples were previously characterized by other molecular testing methodologies. Concordance was analyzed and discordance was addressed by an orthogonal technology.

MATERIALS AND METHODSThe repository specimen were acquired from various vendors. Quality Control and Proficiency Test samples were purchased from Qnostics (Glasgow, Scotland). Nucleic acids were extracted with MagMAX™ Viral/Pathogen Ultra Nucleic Acid Isolation Kit on King Fisher Flex System (Thermo Fisher Scientific). The extracted DNA/RNA samples were tested for the presence of pathogenic microorganisms on a Respiratory Tract Microbiota (RTM) panel that are spotted on OpenArrayTM plates (Figure 1A) with one step reverse transcription (RT) and preamplification, and then followed by qPCR run on real-time instrument QuantStudio 12K (Figure 1B). The RTM panel covers 42 targets of viral, bacterial and fungal organisms (Table 1). Target detection was compared with the previous characterization. For discordant samples, capillary electrophoresis (CE) sequencing analysis was carried out following standard protocol.

Table 3. High Positive Percent Agreement (PPA) and High Accuracy in RTM Panel Table 5. Representative Samples Showing RTM Detection and Sequencing Verification

TRADEMARKSThe trademarks mentioned herein are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.

RESULTS

The RTM panel covers 42 species, and most of them are from a wide range of viruses, both DNA and RNA viruses. It also includes some bacteria and one fungal targets. This is the most comprehensive respiratory panel currently available in the market.

Table 1. List of Targeted Species in TaqMan® Respiratory Tract Microbiota (RTM) Panel

Fig. 1B RTM Workflow. Nucleic acids were extracted King Fisher Flex System(a). RT-preamplification is performed on GeneAmp PCR system 9700TM (b). PCR mix is set up on OpenArrayTM 384–Well Sample Plate and then is transferred onto OpenArrayTM plate (c) by the QuantStudioTM OpenArrayTM AccuFill™ System (d). The sealed plate is placed on the QuantStudioTM12K Flex system (e) for thermal cycling and data collection.

Fig. 1A QuantStudioTM

12K Flex OpenArrayTM

plate. OpenArrayTM

technology utilizes a microscope slide–sized plate with 3,072 through-holes. Each plate contains 48 subarrays with 64 through-holes. Each through-hole is 300 μm in diameter and 300 μm in depth. Each through-hole is coated with hydrophilic and hydrophobic coatings. Reagents are retained in the through-holes via surface tension

Figure 1. OpenArrayTM Technology and RTM Workflow

Three panels (RespI17, RespII17, and RespIII17) of QCMD samples were used to evaluate accuracy of the RTM panel. The panels consists of both negative and positive controls covering common respiratory pathogens. The RTM panel was able to detect all identities of controls. One control influenza type A H1N1 was missed by influenza A H1 assay, but it was identify correctly by influenza A pan assay.

Table 2. RTM Correctly Identify Proficiency Test Control (QCMD) Samples.Table 4. Sequencing Summary Additional and Original Targets Detected by RTM

1A

* Influenza A H1N1 was detected by influenza A assay but not by influenza A H1 assay.

CONCLUSIONS

1. A large collection of TaqMan® assays have been developed to target respiratory potential pathogens. The Respiratory Tract Microbiota (RTM) panel is the most comprehensive PCR-based respiratory panel that is currently on market.

2. We have evaluated the Respiratory Tract Microbiota (RTM) panel with clinical research repository specimens and QCMD reference samples.

3. High Positive Percent Agreement (PPA) was observed between RTM results and other nucleic acid tests on market.

4. Sanger sequencing was performed on both originally claimed targets and additional targets detected only by RTM and further confirmed the high accuracy of the RTM panel.

5. In conclusion, the performance demonstrates high accuracy in identifying potential respiratory pathogens and provides an effective tool for researchers to understand pathogenicity in respiratory tract infections.

A total of 316 repository samples (333 targets) retrospectively were tested by RTM panels. The RTM results were compared with characterization by other nucleic acid tests in market. Initial PPA is 94.6%. Sanger sequencing was conducted on all the 18 discordant targets - 13 of them support RTM results, 1 showed RTM missed the call, and 4 failed sequencing.

RTM detects more targets than other platforms. A total of 222 Sanger sequencing runs were performed, including additional targets RTM detected and targets originally claimed by other platforms. Except for 50 sequencing attempts that did not generate sequencing results, the 172 sequencing results confirmed 100% of the additional targets and vast majority of the targets claimed by other tests.

Some of the representative samples with Sanger orthogonal verification are shown in Table 5, including the original characterized targets and additional targets detected by the RTM panel. All the original characterization are detected by RTM and confirmed by Sanger sequencing. Almost all additional targets by RTM are confirmed by Sanger sequencing except those with Ct values from RTM are higher than 25-26, which may reach the Sanger sequencing detection limit.

1B

Sample Prep RT Preamplification

Plate Loading and qPCR Data Analysis

(c)

(b) (a) (d) (e)

Control Identity Counts ResultsAdenovirus 1 Detected

Coronavirus - NL63 2 Both Detected

Coronavirus - OC43 1 Detected

Enterovirus 68 1 Detected

Human MPV 3 All Detected

Influenza A (H1N1) 1 Detected*

Influenza A 3 All Detected

Influenza B 2 Both Detected

Parainfluenza 1 1 Detected

RSV A 2 Both Detected

RSV B 2 Both Detected

Rhinovirus 2 Both Detected

Bordetella pertussis 1 Detected

Haemophilus influenzae 2 Both Detected

Legionella pneumophila 2 Both Detected

Mycoplasma pneumoniae 1 Detected

Streptococcus pneumoniae 2 Both Detected

Negative 3 Confirmed

Overall 31 31*

Targets Claimed by Other Tests

Detected by RTM

PPA

Influenza A 4 4 100.0%

Influenza A (H1N1) 7 7 100.0%

Influenza A (H3) 41 41 100.0%

Influenza B 28 24 85.7%

RSV A 37 36 97.3%

RSV B 53 53 100.0%

RSV non-specified 2 2 100.0%

Rhinovirus – enterovirus 66 61 92.4%

HPIV-1 21 17 81.0%

HPIV-2 7 6 85.7%

HPIV-3 38 37 97.4%

HPIV 2 2 100.0%

hMPV 10 9 90.0%

Adenovirus 13 12 92.3%

Human coronaviruses 229E 2 2 100.0%

Human coronaviruses HKU1 1 1 100.0%

Human coronaviruses OC43 1 1 100.0%

Sum 333 315 94.6%

Sequenced TargetsAdditional Claimed

RTM Detected

Sequencing Confirmed

Sequencing Confirmed

RTM Detected

Adenovirus 1 1 1 1CoV_229E - - 2 2Flu_A_H3 3 3 6 6

Flu_A_pan 4 4 2 2Flu_B_pan 1 1 2 1

hPIV1 - - 4 4hPIV3 4 4 7 7RSV A 2 2 4 4RSV B 3 3 6 6

Rhinovirus 19 19 31 31CoV_HKU1 1 1 - -CoV_OC43 1 1 - -CoV_NL63 2 2 - -

hMPV 1 1 - -K. pneumoniae 1 1 - -

H. influenzae 14 14 - -HBoV 4 4 - -HHV4 4 4 - -HHV5 3 3 - -HHV6 11 11 - -

S. aureus 11 11 - -S. pneumoniae 12 12 - -M. catarrhalis 1 1 - -

Measles 1 1 - -P. jirovecii 3 3 - -

Sum 107 107 65 64

SampleIDs

Benchmark Targets

Targets Detected by RTM

Sequencing Status

Sequencing Results

Ct Valueswith RTM

TM05395 Rhino-ent

Rhinovirus Performed Confirmed 25.6Haemophilus influenzae Performed Confirmed 14.2Staphylococcus aureus Performed Confirmed 21.0

Streptococcus pneumoniae Performed Confirmed 22.7Human herpesvirus 6 (HHV6) Performed Confirmed 26.3

Klebsiella pneumoniae Performed Confirmed 18.8

TM05410 Rhino-ent

Rhinovirus Performed Confirmed 20.7Haemophilus influenzae Performed Confirmed 20.5Staphylococcus aureus Performed No Result 27.3

Streptococcus pneumoniae Performed Confirmed 13.1Cytomegalovirus (VMV, HHV-5) Performed Confirmed 18.5

Human bocavirus Performed Confirmed 25.2Klebsiella pneumoniae Not performed n/a 28.0

TM05415 Rhino-ent

Rhinovirus Performed Confirmed 16.8Haemophilus influenzae Performed Confirmed 17.8Staphylococcus aureus Performed Confirmed 15.0

Streptococcus pneumoniae Performed Confirmed 13.7Human bocavirus Performed Confirmed 26.2

Adenovirus Not performed n/a 25.1

TM05417 Rhino-ent

Rhinovirus Performed Confirmed 17.6Human bocavirus Performed Confirmed 19.6K. pneumoniae Not performed n/a 26.0S. pneumoniae Not performed n/a 26.7

TM05407 Adenovirus

Adenovirus Performed Confirmed 7.0Hemophilus influenzae Performed Confirmed 11.0

Streptococcus pneumoniae Performed Confirmed 14.6Cytomegalovirus (VMV, HHV-5) Performed Confirmed 20.8Human herpesvirus 6 (HHV6) Performed Confirmed 24.2

TM05418 Influenza A Influenza Type A Performed Confirmed 23.1Staphylococcus aureus Performed Confirmed 21.4

TM05421 Flu A H3

Flu A H3 Performed Confirmed 24.4Haemophilus influenzae Performed Confirmed 10.7Staphylococcus aureus Performed Confirmed 14.6

Streptococcus pneumoniae Performed Confirmed 15.8

TM05402 Coronavirus 229 EHuman coronavirus 229E Performed Confirmed 11.9Haemophilus influenzae Performed Confirmed 15.9

Streptococcus pneumoniae Performed Confirmed 26.2

TM05409 Coronavirus 229 E

Human coronavirus 229E Performed Confirmed 19.1Staphylococcus aureus Performed Confirmed 22.2

Streptococcus pneumoniae Performed Confirmed 16.1Cytomegalovirus (VMV, HHV-5) Performed No Result 26.7Human herpesvirus 6 (HHV6) Performed No Result 26.8

TM05416 Parainfluenza 3Human parainfluenza virus type 3 Performed Confirmed 17.7

Haemophilus influenzae Performed Confirmed 9.7Human herpesvirus 6 (HHV6) Performed Confirmed 29.9

TM05403 Parainfluenza 3Human parainfluenza virus type 3 Performed Confirmed 21.7

Epstein–Barr virus (HHV4) Performed Confirmed 15.2Human coronaviruses OC43 Not performed n/a 24.7